Monoclonal antibody against estrogen stimulated leucine aminopeptidase

ABSTRACT

The identification and characterization of risk factors and their molecular implications in the pathophysiology of human diseases such as cancer is essential for designing efficient diagnostic assays and therapeutic compounds. Estrogenic steroids, under normal physiological conditions, have been shown to play a critical function in several tissues. The response of such a variety of tissues to estrogen stimulation can explain in part its active role in the development and progression of different human diseases, particularly Breast Cancer. Searching for estrogen-responding cellular factors in parental cells of primary human breast carcinomas obtained from tumour biopsies an isoenzyme of putative Leucine Aminopeptidase (LAPase; EC 3.4.11.1) was identified. Results have demonstrated that this marker is found to be elevated in the sera of women with invasive ductal and metastatic carcinomas. A monoclonal antibody against this cellular marker have been produced. This invention refers to the use of LAPase monoclonal antibodies for first line confirmatory blood-based testing for Breast Cancer.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of application Ser. No.09/538,831, filed Mar. 30, 2000.

[0002] The present invention relates to a monoclonal antibody whichdemonstrates specific binding to human estrogen-responsive isoenzyme ofLeucine Aminopeptidase (es-LAPase). The present invention also relatesto the hybridoma cell line, designated as 7B6, and the monoclonalantibody produced by the same. The present invention further relates toa diagnostic system using the monoclonal antibody from the hybridomacell line 7B6, to detect blood, serum or plasma levels of the estrogenresponsive isoenzyme of Leucine Aminopeptidase. The antibody isparticularly useful for rapid diagnostic tests for breast cancer.

BACKGROUND OF THE INVENTION

[0003] The identification and characterization of risk factors and theirmolecular implications in the pathophysiology of human diseases such asbreast cancer is essential for designing efficient diagnostic assays andtherapeutic compounds. Amongst the various risk factors associated withthe onset of early events leading to Breast Cancer, estrogen andestrogen-like compounds with estrogenic mimicking activity remain themost important determinants in the early events and progression ofbreast carcinogenesis. Under normal physiological conditions, there areseveral tissues whereby estrogenic steroids have been shown to play acritical function. These include the development of the reproductivetract, particularly secondary organs, such as the mammary glands. Inaddition, estrogens are also involved in the fine regulation of bonegrowth, liver and cardiovascular function and the estrus cycle, mostlikely through the induction of cell proliferation in target tissues[Sutherland R. L. et al., pp. 197-215, Elsevier Science Publishing B.V.,Amsterdam., Shekhar P. V. M., et al., J. Natl, Cancer, Inst., 89:1774-1782]. The response of such a variety of tissues to estrogenstimulation can explain in part its active role in the development andprogression of different human carcinomas and in particular of BreastCancer. Although the precise molecular mechanisms by which estrogenstimulation regulates various physiological functions requires furtherelucidation, this steroid is involved in both “immediate-early” and“early” events of cell function. In this regard, it appears thatimmediate early events induced by estrogen lead to an increased cellularproliferation most likely through the reduction in the cell cycle byaccelerating the rate at which cells progress from the G₁ phase towardsthe S phase. Recently, it has been proposed that estrogen promotescellular proliferation by co-activating at similar estrogenconcentrations, the expression of cyclin Dl-Cdk4 and cyclin E-Cdk2, twocritical and potentially interrelated G₁ regulatory peptides [Prall, O.W. J., et al., J. Biol. Chem., 272: 10882-10894].

[0004] Diagnostic assays are available for breast cancer. For example,imaging techniques such as ultrasounds and x-rays (mammographies) arewidely used to detect tumors. These imaging techniques, however, sufferfrom a limitation in the resolution of the image which prevents thedetection of tumors below a certain size.

[0005] Histological analysis of biopsies is also a common procedure forthe diagnosis of breast cancer and this technique relies onidentification of visible phenotypes of the cells. However, thisanalysis is somewhat subjective and depends on the skill of theexaminer.

[0006] Molecular diagnostics assays are also available. Among the mostwidely relied upon is an assay which measures estrogen and progesteronereceptors on cells obtained from biopsies. This assay is useful todetermine whether a particular type of cancer will be responsive tohormonal therapy.

[0007] Flow cytometry can also be used to measure parameters, such asDNA content and the proportion of cells in a particular phase of thecell cycle, that correlates with the presence of cancerous cells.

[0008] However, histology, estrogen and progesterone receptor analysisand flow cytometry all require that biopsies be taken and that thesample be extensively processed.

[0009] Therefore, there is a need for more rapid and less invasivemethods of diagnosing breast cancer. In particular, sensitive assays todetect the presence of cellular markers, preferably in the blood, whichreflect the presence of metastasis are desirable. Even more desirable,given the prominent role of estrogen in the progression of breastcancer, are cellular markers, other than estrogen and progesteronereceptors, responsive to estrogen.

SUMMARY OF THE INVENTION

[0010] The present invention relates to a monoclonal antibody whichdemonstrates specific binding to human estrogen-stimulated leucineaminopeptidase (es-LAPase). The present invention further relates to adiagnostic system using the monoclonal antibody to detect blood, serum,plasma or tissue levels of the es-LAPase.

[0011] Thus, according to the present invention there is provided amonoclonal antibody which is specific for es-LAPase.

[0012] In a further embodiment of the present invention there isprovided a method of detecting breast cancer in a patient by determiningthe level of es-LAPase in a sample.

[0013] This invention is also directed to a method for detecting ametastatic cancer in a patient by determining the level of es-LAPase ina sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] These and other features of the invention will become moreapparent from the following description in which reference is made tothe appended drawings wherein:

[0015]FIG. 1 shows the effect of estrogen on LAPase activity in cellsupernatants of primary parental breast cancer cell lines.

[0016]FIG. 2 shows the kinetics of inhibition of LAPase by Bestatin,thioredoxin and glutathione.

DESCRIPTION OF PREFERRED EMBODIMENT

[0017] The present invention relates to a monoclonal antibody whichdemonstrates specific binding to human estrogen-stimulated leucineaminopeptidase (es-LAPase) either in its soluble or membrane associatedform. The present invention further relates to a diagnostic system usingthe monoclonal antibody to detect blood, serum, plasma or tissue levelsof the es-LAPase.

[0018] Searching for estrogen-responding cellular factors in parentalcells of primary human breast carcinomas obtained from tumour biopsies,the inventor isolated an isoenzyme of the putative leucineaminopeptidase (LAPase; EC 3.4.11.1). This LAPase isoenzyme is releasedinto the extracellular environment upon estradiol incubation of parentalepithelial-like cells from human breast carcinomas. Estrogen activationwas dose-dependent with an optimal LAPase induced activation at 100 nM.

[0019] To obtain a leucine aminopeptidase isoenzyme responsive toestrogens, epithelial-like cells from human breast carcinoma biopsieswere incubated with an estrogen. More preferably with estradiol atconcentrations of between 10⁻⁸M and b 10 ⁻⁵M and preferably at aconcentration of 10⁻⁷M. With reference to FIG. 1 it is observed thatthis incubation promotes the release of es-LAPase. The enzyme waspurified from cell supernatants by HPLC-gel permeation followed byDEAE-Cellulose and Bestatin-Sepharose affinity chromatography. Thepurified es-LAPase exhibits a molecular weight of 315 kDa as estimatedby gel permeation on a Bio-Sil™ SEC-250 column (600×7.5 mm) usingthyroglobulin (MW 670 kDa); bovine gamma globulin (MW 18 kDa); chickenovalbumin (MW 44 kDa) and equine myglobin (17 kDa). This isoenzyme ofLAPase of the present invention can thus be distinguished from otherisoenzymes reported in the literature and which exhibit differentmolecular weights.

[0020] The monoclonal antibody of the present invention was prepared byconventional procedures, generally following the methods of Campbell(Campbell, A. M. (1984). Amsterdam, Elsevier: 219-223.) and Lietzke andUnsicker (Lietzke, R. et al., (1985) J. Immunol. Methods, 76:223-228).According to this method, tissue culture adapted mouse myeloma cells arefused to antibody producing cells from immunized mice to obtain hybridcells that produce large amounts of a single antibody molecule. Ingeneral, the antibody producing cells are prepared by immunizing ananimal, for example, mouse, rat, rabbit, sheep, horse, or bovine, withan antigen. The immunization schedule and the concentration of theantigen in suspension is such as to provide useful quantities ofsuitably primed antibody producing cells. These antibody producing cellscan be either spleen cells, thymocytes, lymph node cells and/orperipheral blood lymphocytes.

[0021] The antibody producing cells are then fused with myeloma cells,cell lines originating from various animals such as mice, rats, rabbits,and humans can be used, using a suitable fusion promoter. Many mousemyeloma cell lines are known and available generally from members of theacademic community and various depositories, such as the American TypeCulture Collection, Manassas, Va. The myeloma cell line used shouldpreferably be medium sensitive so that unfused myeloma cells will notsurvive in a selective media, while hybrids will survive. The cell linemost commonly used is an 8-azaguanine resistant cell line, which lacksthe enzyme hypoxanthine-guanine-phosphoribosyl-transferase and thereforewill not be supported by HAT (hypoxanthine-aminopterin-thymidine)medium. In general, the cell line is also preferably a “non-secretor”type, in that it does not produce any antibody. The preferred fusionpromoter is polyethyleneglycol having an average molecular weight fromabout 1000 to about 4000. Other fusion promoters such aspolyvinylalcohol, a virus or an electrical field can also be used.

[0022] The immortalized cells (hybridoma) must then be screened forthose which secrete antibody of the correct specificity. The initialscreening is generally carried out using an enzyme-linked immunosorbentassay (ELISA). Specifically, the hybridoma culture supernatants areadded to microtitre plates which have been previously coated with theantigen, in this case purified es-LAPase. A bound specific antibody fromthe culture supernatants can be detected using a labelled secondantibody, for example, goat antimouse IgG labelled with peroxidase,which is commercially available. Cultures that are positive againstes-LAPase antigen are then subjected to cloning by the limiting dilutionmethod. Secondary hybridoma cultures are re-screened as described above.The cultures are then evaluated as to determine whether or not theantibody binds the antigen and to determine the kinetic profile ofantigen binding. Selected cultures based on these results are subject tofurther cloning until culture stability and clonality are obtained.Immediately after hybridization, the fusion products will haveapproximately 80 chromosomes, and as these cells proceed to divide theywill randomly lose some of these chromosomes. The cloning process is toselect those cells which still have the chromosomes coding for antibodyproduction. The cloning process is repeated until 100% of thesub-population exhibits the production of a specific antibody, which isindicative of the “stability” of the hybridoma. In addition, hybridomaculture wells often have multiple colonies some of which may be antibodynon-producers. The cloning process allows the selection of a positivehybrid which is derived from a single cell.

[0023] In one embodiment of the present invention there is provided amonoclonal antibody, which has been designated MAb 7B6. The hybridomalcell line producing this monoclonal antibody has been deposited with theInternational Depositary Authority of Canada, Room 5190, 1015 ArlingtonStreet, Winnipeg, Manitoba, Canada, R3E 3R2 on Mar. 23, 2000, underAccession Number IDAC 230300-1. This monoclonal antibody is of the IgGla subtype. It recognizes both the soluble and membrane associated formof es-LAPase. A person of ordinary skill in the art will recognize thatother antibodies, both polyclonal and monoclonal can be preparedaccording to the present invention. In one aspect of this invention, MAb7B6 can be coupled to a solid matrix. The matrix may be, but is notlimited to, a protein G matrix. MAb 7B6 coupled to a matrix may be usedto immunoprecipitate es-LAPase. This immunoprecipitation may reducenon-selective binding of the antibody. The immunoprecipitate may beuseful to determine the activity of the enzyme or to perform otherassays.

[0024] The hybridoma cell lines described in the present applicationhave been deposited in accordance with 37 C.F.R. §1.808. Furthermore,subject to paragraph (b) of 37 C.F.R. §1.808, all restrictions imposedby the depositor on the availabiity to the public of the depositedmaterial will be irrevocably removed upon the granting of any patentissuing from this application or from any continuing application basedthereon.

[0025] The present invention also encompasses the antibody of thepresent invention and any fragments thereof containing the activebinding region of the antibody such as Fab, F(ab)₂ and Fv fragments.These fragments can be obtained from the 7B6 antibody by usingtechniques well known to those of skills in the art (Rousseaux et al.Methods Enzymology, 121:663-69, Academic Press, 1986).

[0026] A further embodiment of the present invention encompassesantibodies or fragments thereof capable of binding the same antigenicdeterminant as the 7B6 antibody. Including, but not limited to,antibodies possessing the same antigenic specificity as the 7B6 antibodybut originating from a different species or having a different isotypeor exhibiting different binding affinities. It is envisioned that classand isotype variants of the antibody of the present invention can beprepared using recombinant class switching and fusion techniques thatare well known to those skilled in the art (see for example: Thammana etal. Eur. J. Immunol, 13:614, 1983; Oi et al., Biotechnologies,4(3):214-221, Liu et al. Proc. Nat'l. Acad. Sci. (USA), 84:3439-43,1987; Neuberger et al., Nature 312:604-608, 1984 and Spira et al. J.Immunol. Meth., 74:307-15, 1984).

[0027] The monoclonal antibody of the present invention can be producedeither using a bioreactor or from ascites, both procedures of which arewell known in the art.

[0028] The monoclonal antibody of the present invention can be used inan immunoassay system for determining blood, serum, plasma or tissuelevels of es-LAPase.

[0029] Current immunoassays utilize a double antibody method fordetecting the presence of an analyte. These techniques are reviewed in“Basic Principals of Antigen-Antibody Reaction”, Elvin A. Labat,(Methods in Enzymology, 70, 3-70, 1980). Such systems are often referredto as fast format systems because they are adapted to rapiddeterminations of the presence of an analyte. The system requires highaffinity between the antibody and the analyte. According to oneembodiment of the present invention, the presence of es-LAPase isdetermined using a pair of antibodies, each specific for es-LAPase. Oneof said pairs of antibodies is referred to herein as a “detectorantibody” and the other of said pair of antibodies is referred to hereinas a “capture antibody”. The monoclonal antibody of the presentinvention can be used as either a capture antibody or a detectorantibody. The monoclonal antibody of the present invention can also beused as both capture and detector antibody, together in a single assay.One embodiment of the present invention thus uses the double antibodysandwich method for detecting es-LAPase in a sample of biological fluid.In this method, the analyte (es-LAPase) is sandwiched between thedetector antibody and the capture antibody, the capture antibody beingirreversibly immobilized onto a solid support. The detector antibodywould contain a detectable label, in order to identify the presence ofthe antibody-analyte sandwich and thus the presence of the analyte.

[0030] Common early forms of solid supports were plates, tubes or beadsof polystyrene which are well known in the field of radioimmunoassay andenzyme immunoassay. More recently, a number of porous material such asnylon, nitrocellulose, cellulose acetate, glass fibres and other porouspolymers have been employed as solid supports.

[0031] One embodiment of the present invention uses a flow-through typeimmunoassay device. Valkirs et al. (U.S. Pat. No. 4,632,901) discloses adevice comprising antibody, specific to an antigen analyte, bound to aporous membrane or filter to which is added a liquid sample. As theliquid flows through the membrane, target analytes bind to the antibody.The addition of the sample is followed by the addition of a labelledantibody. The visual detection of the labelled antibody provides anindication of the presence of the target analyte in the sample.

[0032] Another example of a flow-through device is disclosed in Kromeret al. (EP-A 0 229 359), which described a reagent delivery systemcomprising a matrix saturated with a reagent or components thereofdispersed in a water soluble polymer for controlling the dissolutionrate of the reagent for delivery to a reaction matrix positioned belowthe matrix.

[0033] In migration type assays, a membrane is impregnated with thereagents needed to perform the assay. An analyte detection zone isprovided in which labelled analyte is bound and assay indicia is read.For example, see Tom et al. (U.S. Pat. No. 4,366,241), and Zuk (EP-A 0143 574). Migration assay devices usually incorporate within themreagents which have been attached to coloured labels thereby permittingvisible detection of the assay results without addition of furthersubstances. See for example Bernstein (U.S. Pat. No. 4,770,853), May etal. (WO 88/08534), and Ching et al. (EP-A 0 299 428). The monoclonalantibody of the present invention can be used in all of these knowntypes of flow-through devices.

[0034] Direct labels are one example of labels which can be usedaccording to the present invention. A direct label has been defined asan entity, which in its natural state, is readily visible, either to thenaked eye, or with the aid of an optical filter and/or appliedstimulation, e.g. U.V. light to promote fluorescence. Among examples ofcoloured labels, which can be used according to the present invention,include metallic sol particles, for example, gold sol particles such asthose described by Leuvering (U.S. Pat. No. 4,313,734); dye soleparticles such as described by Gribnau et al. (U.S. Pat. No. 4,373,932)and May et al. (WO 88/08534); dyed latex such as described by May,supra, Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated inliposomes as described by Campbell et al. (U.S. Pat. No. 4,703,017).Other direct labels include a radionucleotide, a fluorescent moiety or aluminescent moiety. In addition to these direct labelling devices,indirect labels comprising enzymes can also be used according to thepresent invention. Various types of enzyme linked immunoassays are wellknown in the art, for example, alkaline phosphatase and horseradishperoxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactatedehydrogenase, urease, these and others have been discussed in detail byEva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods inEnzymology, 70. 419-439, 1980 and in U.S. Pat. No. 4,857,453.

[0035] Other examples of biological diagnostic devices, which can beused for the detection of es-LAPase, using the monoclonal antibody ofthe present invention, include the devices described by G. Grenner, P.B. Diagnostics Systems, Inc., in U.S. Pat. Nos. 4,906,439 and 4,918,025.

[0036] In one embodiment of the present invention, the diagnostic testuses a blood sample tube which is commonly used to draw blood samplesfrom patients. The inside wall of the tube acts as a carrier for themonoclonal or polyclonal antibodies and required reagents or detectionmeans, needed to produce a measurable signal. In this embodiment thecapture antibody is immobilized onto the wall of the test tube. Afterthe sample is drawn from the patient, the user simply shakes the samplewith the detector antibody in the tube so that the detector antibodyreacts with any es-LAPase in the blood. In this example the monoclonalantibody of the present invention can be either the capture antibody orthe detector antibody. It may be necessary to use a sample wherein thered blood cells have been removed, so that the red blood cells will notinterfere with the analysis of the results. If the analyte is present inthe blood, it will be sandwiched between the capture antibody and thedetector antibody which contains a suitable label for direct detectionor reacts with the reagents in an indirect assay. The solid support (thetest tube) can then be rinsed free of unbound labelled material. Avariety of solid supports can be used according to this method, forexample, test tube walls, plastic cups, beads, plastic balls andcylinders including microtitre plates, paper, and glass fibres.

[0037] There are currently available several types of automated assayapparatus which can undertake rapid format assays on a number of samplescontemporaneously. These automated assay apparatus includecontinuous/random access assay apparatus. Examples of such systemsinclude OPUS™ of PB Diagnostic System, Inc. and the IMX™ Analyzerintroduced by Abbott Laboratories of North Chicago, Ill. in 1988. Ingeneral, a sample of the test fluid is typically provided in a samplecup and all the process steps including pipetting of the sample into theassay test element, incubation and reading of the signal obtained arecarried out automatically. The automated assay systems generally includea series of work stations each of which performs one of the steps in thetest procedure. The assay element may be transported from one workstation to the next by various means such as a carousel or movable rackto enable the test steps to be accomplished sequentially. The assayelements may also include reservoirs for storing reagents, mixingfluids, diluting samples, etc. The assay elements also include anopening to permit administration of a predetermined amount of a samplefluid, and if necessary, any other required reagent to a porous member.The sample element may also include a window to allow a signal obtainedas a result of the process steps, typically a fluorescent or acolorimetric change in the reagents present on the porous member to beread, such as by a means of a spectroscopy or fluorometer which areincluded within the assay system.

[0038] The automated assay instruments of PB Diagnostic Systems, Inc.are described in U.S. Pat. Nos. 5,051,237; 5,138,868; 5,141,871 and5,147,609.

[0039] A description of the IMX™ Analyzer is included in the “Abbott IMXAutomated Bench Top Immunochemistry Analyzer System” by Fiore, M. etal., Clinical Chemistry, 35, No. 9, 1988. A further example of theseanalyzers has been described in U.S. Pat. No. 4,956,148 entitled“Locking Rack and Disposable Sample Cartridge” issued to C. J. Grandoneon Sep. 1, 1990, and assigned to Abbott Laboratories, which describes acarousel for carrying a plurality of reaction cells for use inconnection with the Abbott IMX™ system. A further development in the arthas been described in Canadian Patent Application 2,069,531, Chadwick M.Dunn et al., assigned to Abbott Laboratories wherein the immunochemistryanalyzer system, described in this prior art application, has thecapability of testing for up to three or four analytes in a single batchduring a single run using currently available instrumentation. Thesystem described in the Canadian application referred to above enablesthe users to group three small batches of assays together rather thanrun three separate analysis. The monoclonal antibody of the presentinvention can be used in these automated analyzers.

[0040] A further class of immunochemical analyzer systems, in which themonoclonal antibody of the present invention can be used, are thebiosensors or optical immunosensor systems. In general an opticalbiosensor is a device which uses optical principles quantitatively toconvert chemical or biochemical concentrations or activities of interestinto electrical signals. These systems can be grouped into four majorcategories: reflection techniques; surface plasmon resonance; fibreoptic techniques and integrated optic devices. Reflection techniquesinclude ellipsometry, multiple integral reflection spectroscopy, andfluorescent capillary fill devices. Fibre-optic techniques includeevanescent field fluorescence, optical fibre capillary tube, and fibreoptic fluorescence sensors. Integrated optic devices include planerevanescent field fluorescence, input grading coupler immunosensor,Mach-Zehnder interferometer, Hartman interferometer and differenceinterfermoter sensors. These examples of optical immunosensors aredescribed in general in a review article by G. A. Robins (Advances inBiosensors), Vol. 1, pp. 229-256, 1991. More specific description ofthese devices are found for example in U.S. Pat. Nos. 4,810,658;4,978,503; 5,186,897; R. A. Brady et al. (Phil. Trans. R. Soc. Land. B316, 143-160, 1987) and G. A. Robinson et al. (in Sensors and Actuators,Elsevier, 1992).

[0041] Another immunochemical analyzer is flow cytometry. In flowcytometry the sample containing the antigen is reacted with afluorescently labelled form of the monoclonal antibody of the presentinvention. The sample is passed in front of a laser beam of a givenwavelength capable of exciting the chromophore on the antibody. Eachparticle or cell having the antibody bound to it will fluoresce and willbe detected. This technique allows the analysis of specific cell typesand in particular of specific blood cell types. It is therefore usefulfor the detection of cells exhibiting the es-LAPase antigen.

[0042] In one embodiment of the present invention, es-LAPase is detectedin a sample of blood, serum or plasma, using the monoclonal antibody ofthe present invention, in a device comprising a filter membrane or solidsupport with a detection section and a capture section. The detectorsection contains an antibody (a detector antibody), which will reactwith es-LAPase. The detector antibody is reversibly immobilized onto thesolid support and will migrate with the sample, when in use. It ispreferred that the detector antibody is labelled, for example with aradionucleotide, an enzyme, a fluorescent moiety, luminescent moiety ora coloured label such as those described in the prior art, and discussedabove. The capture section comprises a capture antibody, which isirreversibly immobilized onto the solid support. The antibodies, captureand detector antibody, and the necessary reagents are immobilized ontothe solid support using standard art recognized techniques, as disclosedin the flow-through type immunoassay devices discussed previously. Ingeneral, the antibodies are absorbed onto the solid supports as a resultof hydrophobic interactions between non-polar protein substructures andnon-polar support matrix material.

[0043] According to this embodiment of the present invention, ifes-LAPase is present in the blood, it will react with the detectorantibody in the detector section and will migrate on the filter membranetowards the capture section where the analyte will further bind with thecapture antibody. Thus, es-LAPase will be sandwiched between the captureantibody and the detector antibody, which contains a suitable label.

[0044] In this example of the present invention, if the detectorantibody is labelled with a coloured label or an enzyme which willproduce a coloured label, the patient's blood would first requirecentrifugation or some pre-filtering in order to remove the red bloodcells so that the colour of the red blood cells will not interfere withthe coloured labels. If radioactive labels or florescent labels are tobe used, a pre-filtration or centrifugation step may not be required. Inthis embodiment, the monoclonal antibody of the present invention can beeither the capture antibody or the detector antibody. In one embodiment,the monoclonal antibody of the present invention is a capture antibody.The detector antibody can be other es-LAPase monoclonal antibodies,monoclonal antibodies reactive to other isoforms of LAPase, orpolyclonal anti-es-LAPase antibodies. Either chicken, rabbit, goat ormouse polyclonal antibodies can be used. Many such antibodies are knownand can be prepared and labelled by known methods.

[0045] This immunoassay system is generally described in U.S. Pat. No.5,290,678. The antibody of this invention is particularly useful in thissystem because of its high affinity for es-LAPase.

[0046] In a further embodiment of this invention the monoclonal antibody7B6 can also be used to monitor patients that are at risk of developingbreast cancer since serum es-LAPase is affected by estrogens and thatestrogens have been identified as important factors in the progressionof certain types of breast cancer. Individual at risk may include thosetaking estrogen based birth control pills, hormonal replacement therapyor individuals having abnormal hormonal patterns.

[0047] As would be recognized by one of skill in the art, the abovedescribed embodiments of this invention may have to be modified todistinguish between the soluble and membrane associated form ofes-LAPase.

[0048] As would also be recognized by one of skill in the art, a baseline level of es-LAPase may be present in normal patients. Thus, in thepresent invention, in certain embodiments, the levels of es-LAPase abovenormal will be determined. This can be accomplished by either comparingthe results to the results of a normal patient, or adjusting thesensitivity of the immunoassay so that only values above a certainthreshold will show as a positive result.

[0049] As will be clearly indicated in the examples infra, es-LAPaselevels are correlated with breast cancer and the presence of metastasisin patients with breast cancer. Thus, the monoclonal antibody 7B6 of thepresent invention, along with the embodiments described supra isparticularly useful as a new diagnostic tool.

[0050] In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting the scope of this invention in anymanner.

EXAMPLES Example 1

[0051] Isolation and Purification of Estradiol-Dependent Leucine AminoPeptidase (LAPSE).

[0052] Primary parental breast carcinoma cells obtained from human tumorbiopsies were stimulated with 100 nM 17-β-Estradiol for 24 hours or cellmedia alone as a control. The cell media was RPMI 1640 medium+10%FCS+100 U/ml Penicillin+100 μg/ml Streptomycin. Cell supernatants werecollected then after and dialyzed against PBS in seamless cellulosetubing (MW 12,400) for 12 hours at 4° C. LAP was subsequently purifiedfrom the dialyzed cell supernatants using HPLC-gel permeation followedby DEAE-Cellulose and Bestatin-Sepharose affinity Chromatography.Briefly, the cell supernatant was applied to a Bio-Sil SEC-250 column(600×7.5 mm) previously equilibrated in a buffer containing 100 mMSodium Phosphate buffer pH 6.8, 100 mM Na₂SO₄, 1 μM ZnCl₂ and 10%glycerol. The column was washed with 300 ml of the same buffer at a flowrate of 0.5 ml/min. Protein was concentrated to 10 ml by ultrafiltrationusing YM5 membrane (5000 M.W. cutoff, Amicon Div., Danvers, Mass., USA).The concentrate was applied to a DEAE cellulose column (2.6 cm×28.5 cm)equilibrated and washed with 50 mM Tris-HCl buffer pH 7.5; 1 μM ZnCl₂;and 10% (v/v) glycerol. es-LAPase was eluted using a linear gradient (0to 1M NaCl in Tris buffer) at a flow rate of 0.50 ml/min. ABestatin-affinity column was prepared using Ultralink EDC/DADPA Amidebonding matrix (Pierce, Rockford, Ill. U.S.A.) by reacting 100 mg ofpure Bestatin with the carbodiimide EDC/DADPA matrix following theprocedure provided by the manufacturer. Prior to loading the es-LAPasecontaining eluent, the Bestatin-affinity column was equilibrated with 10mM Tris-HCl pH 8.0 containing 1 μM ZnCl₂ and washed with 300 ml of thisbinding buffer. es-LAPase was recirculated through the system using aperistaltic pump at a flow rate of 0.10 ml/min, for 2 hours. Followingthis recirculation, the column was washed with eight column volumes ofbinding buffer. Bestatin-bound es-LAPase was eluted with a lineargradient (0-0.5 M NaCl) prepared in binding buffer 10 mM Tris-HCl pH 8.0containing 1 μM ZnCl₂. Elution of bound es-LAPase was monitored byabsorbance at 280 nm. Purified es-LAPase fractions were aliquoted in 500μl and stored until further use in 50 mM Tris-HCl pH 7.8 and 50 μMZnCl₂. es-LAPase protein concentration following each purification stepwas determined as described by Pulido-Cejudo [J. Chromatogr. B 660(1994) 37-47)]. Briefly, samples (100-200 μl) were dialysed againstdeionized water and 2-20 μl of each were placed in polypropylene tubes.Samples were dried for 60 min at 110° C. for 90 min and subsequentlyneutralized with 250 μl of glacial acetic acid. The samples were thenreacted with 500 μl of the following ninhydrin-hydrindantin solution: 2g of ninhydrin and 150 mg of hydrindantin (Sigma) were dissolved in 65ml of 2-methoxyethanol and then 35 ml of 4 M sodium acetate (pH 5.5)were added. The tubes were incubated at 110° C. for 15 min. Beforereading the absorbance of the samples at 570 nm, 2.5 ml of 5 % (v/v)ethanol were added. Protein content was determined by interpolation onan absorbance curve obtained with samples of BSA (1-10 μg). A summary ofthe purification fold of the 17-β-Estradiol-stimulated LAPase fromparental cells of human breast carcinomas is set out in Table 1. TABLE 1Summary of Purification of LAP from Human Breast Carcinoma ParentalCells Specific Total Activity² Protein1 Activity² (nmole/ Step (mg)(nmole/min) min/mg) Fold Yield % Supernatant 15.4 98.21 6.37 1 100 Gel4.2 82.13 19.55 3.07 83.63 Permeation Cellulose 0.72 48.25 67.01 10.5149.13 DEAE Bestatin- 0.005 37.11 7422 1165 37.79 Sepharose

Example 2

[0053] A) Monoclonal Antibody Production and Purification

[0054] In producing the hybridoma cell line 7B6 secreting the mousemonoclonal antibody to 17-β-Estradiol-stimulated LAPase, protocols forantigen preparation for immunization, preparation of spleen cells fromimmune animals, fusion of spleen cells with myeloma cells and plating offused cells in selective medium was conducted following detailedguidelines described by Campbell [Burdon R H, Knippenberg PHV (eds):Laboratory Techniques in Biochemistry and Molecular Biology, Amsterdam,Elsevier, p219 (1984)] and by Lietzke and Unsicker [Leitzke R. UnsickerK: A Statistical Approach to Determine Monoclonality After Limiting CellPlating of a Hybridoma Clone, J Immunol Methods 76:223 (1985)].

[0055] Briefly, the primary immunization was performed with purifiedes-LAPase following desalting. Boosts with purified es-LAPase wereperformed at days 14, 35 & 56. BALB/c mice were screened at days 24 &45. The mice were sacrificed at day 59 and the splenocytes from the bestresponder were fused with myeloma cells. Screening was performed by dotblot immunostaining on nitrocellulose.

[0056] The hybridoma clone 7B6 was obtained by single cell cloning bylimiting dilution. Four dilution tubes in series containing hybridomacells with medium supplement with 20% FBS+2×OPI were prepared. 100 μl ofeach dilution was plated in a 96-well plate with 50 μl of splenocytefeeder cells in each well and placed inside a 37° C. 5% CO₂ incubator.At day 7, supernatants from each well were removed and screened by dotblot immunostaining on nitrocellulose.

[0057] B) Expansion of Hybridoma Clone 7B6

[0058] Hybridoma clone 7B6 cells were transferred from the 96 well plateto 0.5 ml medium supplemented with 20% FBS+1×OPI+1×HAT in a 24 wellplate. Once the cells were dense, they were transferred into 5 mls in a60 mm dish and then transferred to 10 mls in a 100 mm dish. Once in the60 mm dish, the cells were weaned off hypoxanthine, thymidine andaminopterin. 7B6 hybridoma cells were continued to be grown until in alog phase of growth. Anti-es-LAPase, MAb 7B6 was isolated from collectedhybridoma 7B6 cell supernatant by affinity chromatography usingImmunopure IgG as per described by manufacturer. Screening was performedby dot blot immunostaining on nitrocellulose.

[0059] C) Immunotyping of MAb 7B6

[0060] The isotype of MAb 7B6 was determined using Sigma's ImmunotypeKit. Briefly, the assay involves binding of MAb 7B6 to a precoatedisotyping nitrocellulose membrane strip followed by immunodetectionusing a sensitive biotin-avidin-enzyme detection system. Theimmunoglobulin isotype is revealed by self description.

[0061] D) Immobilization of MAb 7B6 to Protein G Matrix.

[0062] Following purification of the MAb 7B6, the corresponding IgG1aisotype was subsequently immobilized though a DSS cross-linking systemobtained from Pierce (Rockford, Ill., U.S.A) according to the proceduresdescribed by the manufacturer. MAb 7 B6 Protein G matrices were used toselectively immunoprecipitate LAPase activity from plasma or cellmembrane-bound fractions prior to determining LAPase actvity.

Example 3 ELISA Analysis of Human Breast Carcinoma Parental Cell Line

[0063] ELISA analysis of human breast carcinoma parental cell lines wasconducted to demonstrate the reactivity of MAb 7B6 against human LAP.Briefly, 50000 parental cells were plated per well in a 96 well plate inRPMI 1640 medium+10% FCS+100 U/ml Penicillin+100 μg/ml Streptomycin. Theplated cells were cultivated at 37° C. 5% CO₂ for 24 hours. The cellsupernatants were removed, the cells were washed with PBS andsubsequently fixed with 1% gluteraldehyde in PBS for 1 hour at roomtemperature. Washing with PBS occurred prior to blocking with casein for1 hour at 37° C. 5% CO₂. Following another wash with PBS, serialdilutions of MAb 7B6 were added to the wells and allowed to incubate for2 hours at 37° C. 5% CO₂ Demonstration of the reactivity of MAb 7B6 wasevident upon the addition of a secondary antibody, anti-(IgG+IgM)peroxidase conjugated goat anti-mouse IgG+IgM (H+L) followed by thesubstrate, OPD. A summary of the readings taken at 490 nm are set out inTable 2. TABLE 2 Summary of Reactivity of MAb 7B6 against LAP from HumanBreast Carcinoma Parental Cells MAb 7B6 Cell Line 1 OD 490 nm − CellLine 2 OD 490 nm − (ng/well) Blank OD 490 nm Blank OD 490 nm 200 0.707 −0.054 = 0.653 0.6 − 0.005 = 0.595 100 0.57 − 0.021 = 0.549 0.446 − 0.003= 0.443 50 0.489 − 0.042 = 0.447 0.327 − 0.003 = 0.324 25 0.38 − 0.047 =0.333 0.24 − 0    = 0.24 12.5 0.294 − 0.05  = 0.244 0.165 − 0    = 0.1656.25 0.226 − 0.05  = 0.176 0.1 − 0.003 = 0.097 3.125 0.155 − 0.044 =0.111 0.068 − 0.003 = 0.065 1.56 0.107 − 0.05  = 0.057 0.043 − 0.002 =0.041 0.78 0.094 − 0.055 = 0.039 0.023 − 0.001 = 0.022 0.39 0.067 −0.073 = 0 0.015 − 0    = 0.015 0.2 0.061 − 0.052 = 0.009 0.008 − 0    =0.008 0.1 0.053 − 0.046 = 0.007 0 − 0    = 0

Example 4

[0064] LAPase Levels in Cell Supernatants Following EstrogenStimulation.

[0065] Primary parental breast carcinoma cells were incubated with 100nM 17-β-Estradiol for 24 hrs and cell media alone as a control. LAPasein supernatants was isolated using MAb 7B6_((IgG1a))-protein-G-LAPasebeads prepared by covalently cross-linking the monoclonal antibody.LAPase activity of the LAPase-immunoprecipitated supernatants wasdetermined fluorometrically using leucine-β-naphthylamide as thesubstrate as described by Kuramochi et al. [Kuramochi, H. et al., (1987)J. Antibiot., 401605-1611].

[0066] Purified preparations of the enzyme obtained from Sigma wereincubated with 100 nM 17-β-Estradiol for 24 hrs. LAPase activity wasdetermined fluorometrically using leucine-β-naphthylamide as thesubstrate as described by Kuramochi et al. [Kuramochi, H. et al., (1987)J. Antibiot., 401605-1611].

[0067] Following estrogen stimulation, maximum release of LAPase wasobserved at an estrogen concentration of 100 nM following 24 hours ofincubation (see FIG. 1). During the same period, LAPase in cellsupernatants of control cells remain unchanged. In addition, LAPaseactivity was determined in supernatants of primary parental cell linesimmunoprecipitated with MAb 7B6_((IgG1a))-protein-G-LAPase matrix boundantibodies. These results show that the extracellular LAPase activity ofestrogen stimulated cells (100 nM) was 7.7×10⁻⁵ U/ml in comparison to6.4×10⁻⁶ U/ml detected in the supernatant of parental cells incubatedfor 24 hours with cell media alone as control. In addition, there was noeffect of estrogen incubation on LAPase activity in purifiedpreparations of this enzyme alone. Collectively, these results suggestthat estrogen effect in LAPase activity encompasses a cellular mediatedprocess.

Example 5

[0068] Estrogen-Responsive LAPase in Women with Breast Cancer.

[0069] MAb 7B6 binding to parental cells detected by immunoflowcytometrycan be used to detect circulating epithelial like-cells in women withBreast Cancer. In addition, plasma immunoprecipitates using MAb 7B6consistently show higher levels of LAPase in women with non-invasiveductal and metastatic carcinomas when compared to plasma levels fromotherwise healthy women.

[0070] A) LAPase Actvity in Plasma of Women with Primary Breast Cancer

[0071] LAPase activity in plasma of relapsed patients with primaryBreast Cancer compared to aged matched controls of otherwise healthywomen, were determined fluorometrically using leucine-β-naphthylamide asthe substrate as described by Kuramochi et al. The reaction was stoppedby boiling the samples at 100° C. for 10 mins, followed bycentrifugation at 780×g at 4° C. for 10 mins. Values obtained representthe average of LAPase activity determined in triplicate in 16 patientsfrom each test group.

[0072] LAPase activity was determined using plasma immunoprecipitatesobtained by immunoprecipitation using Covalent MAb7B6_((IgG1a))-Protein-G-LAPase matrices. Briefly, Plasma was spun at500×g for 10 min. at 4° C. Plasma was removed by aspiration and spunonce more at 500×g for 5min. at 4° C. The resulting plasma was diluted1:10 with PBS and 800 μl of plasma dilution was added to MAb7B6_((IgG1a))-G-LAPase beads containing 5 μg of antibody in 200 ul ofbeads pre-incubated with blocking buffer [50 mM Tris-HCl; 0.5% non-fatdry milk(NFDM)]. Samples were incubated at room temperature (˜22° C.)for 15 min with constant gentle rotation in 0.5% BSA pre-coatedEppendorf tubes. After incubation samples were spun at 250×gEppendorf-Microfuge at room temperature supernatants removed. The beadscontaining the LAPase activity was washed trice with PBS;0.5% NFDM andfinally resuspended in Calcium-free Hank's solution making up a 600 μlfinal reaction volume and 182 μM 1-leucine-β-naphthylamide. Acorresponding blank without LAPase-Covalent MAb 7B6_((IgG1a))-beadscoated with 0.5% NFDM was used as baseline.

[0073] Using selective immunoprecipitation of plasma incubated withanti-LAPase MAb-Protein G matrix, it was found that LAPase activity inthe plasma of women with metastatic disease was four orders of magnitudehigher than the control population. Such increase in LAPase activity wasless predominant in patients with ductal carcinoma in-situ (DCIS). Asummary the results is provided below in Table 3. TABLE 3 LAPaseactivity levels in plasma of women with Breast Cancer Patient PopulationLAPase Activity (n = 10 per group) (U/ml) Normal 6.16 × 10⁻⁴ ± 0.82 ×10⁻⁴ DCIS (non-invasive) 3.06 × 10⁻¹ ± 0.80 × 10⁻¹ Metastatic(Lung/Brain) 1.73 ± 0.08

[0074] B) Flow Cytometric Detection of Epithelial-Like Breast CarcinomaCells

[0075] Indirect immunofluorescence staining of epithelial-like cellsfrom tumour biopsies was performed by incubating adherent cells withhuman serum pre-adsorbed MAb 7B6 antibodies. Briefly, confluent cellswere washed trice with PBS and incubated at 37° C. with 20 μl of MAb 7B6antibodies (200 μg/ml) or control mouse IgG1 in a final volume of 2 ml.After 20 min. incubation cells were washed trice once more with PBS andincubated with mouse anti-IgG-PE or mouse anti-IgG-FITC conjugtes for 15minutes. Cells were subsequently washed three times with PBS, partiallytrypsinized and analyzed in a flowcytometer equipped with an air-cooledargon ion laser operating at 10 mwatt. Simultaneous excitation of FITCand PE conjugates was achieved by setting the excitation wavelength at488 nm.

[0076] As shown in Table 4, MAb 7B6 can be used to detect membrane-boundLAPase in parental epithelial-like cells isolated from human breastcarcinomas. Screening of LAPase reactivity in circulating cells wasexamined in normal women and compared to those with ductal carcinoma insitu and with metastatic Breast Carcinomas. Briefly whole blood was spunand buffy-coat removed. Cells within the buffy-coat were washed tricewith PBS and incubated with MAb 7B6 [20 μl of MAb 7B6 antibodies (200μg/ml) or control mouse IgG1] in a final volume of 2 ml of PBS;0.5%NFDM. The % of MAb 7B6 cells in this fraction was subsequently estimatedby flowcytometry. TABLE 4 Membrane-bound LAPase in parentalepithelial-like cells isolated from Human Breast Carcinomas PatientPopulation (n = 100 per group) % MAb 7B6 Positive cells Normal 0.1-0.5DCIS (non-invasive)  5-14 Metastatic (Lung/Brain) 18-25

[0077] C) LAPase Levels in Plasma of Women with Primary Breast Cancer

[0078] LAPase levels in plasma of women with primary Breast Cancercompared to aged-matched controls of otherwise healthy women, weredetermined by ELISA using the monoclonal antibody MAb 7B6.

[0079] Plasma immunoprecipitates using MAb 7B6 consistently show higherlevels of LAPase in women with non-invasive ductal and metastaticcarcinomas when compared to plasma levels from otherwise healthy women.A summary of results obtained is set out in Table 5. TABLE 5 LAPaselevels in plasma of women with Breast Cancer Patient Population LAPaseLevels (n = 100 per group) (μg/ml) Normal  5-10 DCIS (non-invasive)450-600 Metastatic (Lung/Brain) 1200-3700

[0080] All cited references and Patents or Patent Applications areincorporated herein by reference.

[0081] The present invention has been defined in terms of certainexamples, which are not to be construed as limiting. The full scope ofthe present invention is defined in the following claims.

Example 6

[0082] Inhibitors of es-LAPase

[0083] Human Thioredoxin was purified from CD₄+T MP6 cells as describedby Rosen et al. [Rosen, A., et al. (1995) Int. Immunol. 7, 625-633] andcompared to purified Thioredoxin activity from E. Coli. purchase fromSIGMA-ALDRICH Canada (Oakville, Ontario Canada).

[0084] Thioredoxin was covalently linked through an amide bond toUltralink EDC/DADPA bonding matrix (Pierce, Rockford, Ill. U.S.A.) byreacting 5 mg of purified Thioredoxin with the carbodiimide EDC/DADPAmatrix following the procedure provided by the manufacturer.

[0085] Measurements of LAPase were performed spectrophotometrically at330 nm to measure product formation of β-naphthylamine, using 182 μM1-leucine-β-naphthylamide as the substrate. The kinetic assays wereperformed using a Spectronic Genesys 5 spectrophotometer from Milton Roy(Rochester, N.Y.). Absorbance was recorded every sixty seconds, over aperiod of 30 min. Enzyme kinetic assays were performed using 8.0×10⁻² Uof es-LAPase in a final reaction volume of 600 μl and triplicatesamples. The reaction mixture contained the following materials added inorder and all kept on ice prior to use: es-LAPase, Calcium-free Hank'ssolution making up a 600 μl final reaction volume and 182 μM1-leucine-β-naphthylmide. A corresponding blank without es-LAPase wasused as baseline. Both of the cuvettes were transferred to thespectrophotometer where the 1-leucine-β-naphthylmide solution was addedlast, marking time zero of the reaction. The inhibition studies werecarried out in the presence of 167 μM Bestatin, 167 μM reducedGlutathione and 17 μM reduced thioredoxin.

[0086]FIG. 2 shows the inhibitory effect of both Bestatin and twothiol-containing peptides, reduced thioredoxin and reduced glutathionerespectively. Es-LAPase showed significant slower rates of reaction bothin the presence of thioredoxin and glutathione in comparison to thatobserved in the control es-LAPase. In addition, es-LAPase reaction rateswere slower in the presence of 17 μM thioredoxin when compared to thoseobserved in samples incubated with 167 μM reduced Glutathione. Inaddition, 167 μM Bestatin clearly inhibits es-LAPase activity. As shownin Table 6, reduced thioredoxin is an effective inhibitor of es-LAPaseactivity. The analysis of the maximum reaction rates (Vmax),Michaelis-Menten (Km) constants and the inhibition constants (Ki) foreach inhibitor as presented in Table 6 reveals significant differencesin the inhibitory properties of each peptide. In this regard, es-LAPaseincubated with Bestatin shows a Vmax value very similar to that observedto es-LAPase alone and a significantly higher Km value. This dataconfirms the competitive nature of es-LAPase inhibition by Bestatin. Incontrast, es-LAPase incubated with reduced thioredoxin leads to asignificantly lower Vmax and Km with an intermediate Ki value whencompared to those obtained for Bestatin and glutathione. Collectivelythese data strongly suggest that reduce thioredoxin inhibits es-LAPasein an uncompetitive fashion while with reduced glutathione es-LAPaseinhibition is noncompetitive. TABLE 6 Bestatin, reduced thioredoxin andglutathione es-LAPase inhibition Constants Vmax Km Ki (10⁻⁶ M/min) (10⁻⁴M) (10⁻⁶ M) LAPase alone 36.8 ± 1.9 1.67 ± 0.14 N/A Bestatin 167 μM 34.0± 3.9 48.7 ± 8.4  6.22 ± 1.61 Thioredoxin 17 μM  5.31 ± 0.02 0.234 ±0.006 3.40 ± 0.58 Glutathione 167 μM 17.2 ± 1.5 1.78 ± 0.25 1.53 ± 0.40

What is claimed is:
 1. A method of detecting breast cancer in a patientby determining the level of es-LAPase in a sample.
 2. The methodaccording to claim 1 wherein the level of es-LAPase is determined by amethod selected from the group consisting of: determining the activityof es-LAPase in a sample, and determining the presence of es-LAPase in asample by an immunoassay.
 3. The method according to claim 2 wherein thepresence of es-LAPase is determined by an immunoassay using a monoclonalantibody which is specific for soluble and membrane associatedes-LAPase.
 4. The method according to claim 3 wherein the monoclonalantibody is produced by hybridoma cell line 7B6, deposited with theInternational Depositary Authority of Canada under Accession number IDAC230300-1.
 5. A method of detecting a metastatic cancer in a patient bydetermining the level of es-LAPase in a sample.
 6. The method accordingto claim 5 wherein the level of es-LAPase is determined by a methodselected from the group consisting of: determining the activity ofes-LAPase in a sample, and determining the presence of es-LAPase in asample by an immunoassay.
 7. The method according to claim 6 wherein thepresence of es-LAPase is determined by an immunoassay using a monoclonalantibody which is specific for soluble and membrane associatedes-LAPase.
 8. The method according to claim 7 wherein the monoclonalantibody is produced by hybridoma cell line 7B6, deposited with theInternational Depositary Authority of Canada under Accession number IDAC230300-1.